Thrombospondin receptor binding peptides

ABSTRACT

Novel short peptides are described that bind to the thrombospondin 1 receptor, which preferably have five amino acid residues which share the tetrapeptide Arg-Val-Ala-Val and have the following sequences: 
     Ile-Arg-Val-Ala-Val [SEQ ID NO:13] and 
     Val-Arg-Val-Ala-Val [SEQ ID NO:14].

This is a DIVISION, of application Ser. No. 08/029,333, filed Mar. 5,1993, now U.S. Pat. No. 5,399,667.

This invention was made with Government support under Grant No. HL4147awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to novel binding peptides and, moreparticularly, to small peptides that bind to the thrombospondin 1receptor.

The interaction of cells with extracellular matrix (ECM) molecules is acomplex process from which cells derive a wealth of information abouttheir environment. This information is processed in a number of waysthat ultimately affect cell motility, shape, proliferation and geneexpression (Hynes, 1992). ECM macromolecules like fibronectin, laminin,vitronectin, and collagen have been shown to mediate cell adhesion, aprocess that includes cell attachment and spreading. Like theseproteins, thrombospondin 1 (TS1) promotes adhesion of a number of normaland transformed cell types (Frazier, 1991; Roberts et al., 1987), afunction which underlies many effects that TS1 exerts in severalbiologically complex systems. These effects include stabilizing plateletaggregation (Leung et al., 1984; Dixit et al., 1985), regulating cellgrowth (Majack et al., 1988; Good et al., 1990), specifying thedifferentiation phenotype of certain cells (Castle et al., 1991), woundhealing (Raugi et al., 1987) and the migration of tumor cells (Tuszynskiet al., 1987) and PMNs (Mansfield et al., 1990). A good example of theregulation of several aspects of cellular behavior by TS1 is theinhibition of angiogenesis in vivo and of endothelial cell migration andproliferation in vitro (Good et al, 1990; Taraboletti et al., 1990).

There are at least 4 TS isogenes, TS1, 2 and 3 (Bornstein et al., 1991;LaBell et al., 1992; Laherty et al., 1992; Vos et al., 1992) andcartilage oligomeric matrix protein or COMP (Oldberg et al., 1992) whoseproducts are related, but decidedly different. Of these, platelet TS(which is pure TS1) is the best characterized, and serves as a prototypefor this growing family. Distinct activities can be assigned to certaindomains. For example, the amino-terminal domain of TS1 induces spreadingof G361 cells while the COOH-terminal cell binding domain (CBD) of TS1promotes haptotaxis and attachment of these cells (Taraboletti et al.,1987; Roberts et al., 1985). TS1 contains at least four domains thatsupport cell attachment: the amino-terminal heparin-binding domain(Murphy-Ullrich et al., 1987), the type 1 repeats of about 60 amino acidresidues containing a common subhexapeptide sequence (Prater et al.,1991), the RGD sequence in the last of the type 3 calcium bindingrepeats (Lawler et al., 1988) and the COOH-terminal ca. 220 residuestermed the “cell-binding” domain (CBD, Kosfeld et al., 1991). Monoclonalantibody (MAb) called C6.7 which binds to this CBD and blocks itsinteraction with cellular receptors was previously described (Dixit etal., 1985). Using this MAb it has been shown that the CBD is essentialfor binding of TS1 to platelets (Dixit et al., 1985), many transformedcells (Varani et al., 1986) and to human melanoma cells (Taraboletti etal., 1987). The CBD of TS1 (rCBD) exclusive of the upstream RGD sequencehas been expressed in bacteria and its attachment activity for humanmelanoma cells has been demonstrated (Kosfeld et al., 1991).

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel small synthetic peptidesare provided that bind to the thrombospondin 1 (TS1) receptor. Thesepeptides preferably have 5-13 amino acid residues which share thetripeptide Val-Val-Met and have the following sequences:

RFYVVMWKQVTQS [SEQ ID NO:1] and fragments thereof containing the minimalsequence RFYVVM [SEQ ID NO:3], and

FIRVVMYEGKK [SEQ ID NO:4] and fragments thereof containing the minimalsequence IRVVM [SEQ ID NO:5].

The novel VVM-containing peptides of the invention are illustrated byfive preferred pep tides in which the sequences are converted to thethree-letter abbreviations and designated herein and in the SequenceListing of the accompanying Diskette as f follows:

Arg Phe Tyr Val Val Met Trp Lys Gln Val Thr Gln Ser [SEQ ID NO:1]                5                   10 Arg Phe Tyr Val Val Met Trp Lys[SEQ ID NO:2]                 5 Arg Phe Tyr Val Val Met [SEQ ID NO:3]                5 Phe Ile Arg Val Val Met Tyr Glu Gly Lys Lys [SEQ IDNO:4]                 5                   10 Ile Arg Val Val Met [SEQ IDNO:5]                     5

The foregoing 5 illustrative peptides are also designated herein forstructural purposes as 4N1, 4N1-1, 4N1-2, 7N3 and 7N3-1, respectively.

The novel binding peptides of this invention are contained in 2non-overlapping 30-residue synthetic peptides, designated C4 and C7,respectively, of the thrombospondin 1 (TS1) COOH-terminal cell bindingdomain (CBD). These novel peptides retain the binding activity of theparent 30-mer peptides and faithfully reflect the binding activity ofCBD.

These results were unexpected in view of the fact that, by way ofdistinction, the following closely related peptides were inactive insaid binding:

Gly-Arg-Val-Val-Met [SEQ ID NO:6],

Ile-Glu-Val-Val-Met [SEQ ID NO:7] and

Ile-Arg-Val-Val-Gly [SEQ ID NO:8].

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand specifically claiming the subject matter regarded as forming thepresent invention, it is believed that the invention will be betterunderstood from the following preferred embodiments of the inventiontaken in conjunction with the accompanying drawings in which:

FIG. 1A shows the amino acid sequences of the subpeptides derived frompeptide C4 of the CBD of TS1. The boxed letters indicate amino acidresidues that are common in all active subpeptides.

FIG. 1B is a graphical representation which shows the direct attachmentof G361 cells to C4 subpeptides. Equimolar concentrations of peptidesshown in FIG. 1A were evaluated as attachment factors for G361 cells.Peptides C4 (¤), 4N (), 4N1 (▴), 4N1-1 (♦), 4N2 (▪) and 4C () wereadsorbed to microtiter plates at the indicated concentrations. Cellswere added to the wells and incubated for 1 hour at 37° C. The attachedcells were quantitated as described under “Materials and Methods”hereinbelow, and the actual absorbance due to endogenous cellularphosphatase hydrolysis of p-Nitrophenyl phosphate at 410 nm is plotted.

FIG. 2A shows the amino acid sequences of subpeptides derived frompeptide C7 of the CBD of TS1. The boxed letters indicate the amino acidresidues that are in common in all active subpeptides.

FIG. 2B is a graphical representation which shows the direct attachmentof G361 cells to subpeptides of peptide C7. Microtiter wells wereadsorbed with peptides C7 (▪), 7N (), 7N3 (▴), 7N3-1 (♦), 7N2 (▪),7N2-2 (∘), 7N3-2 (▴) and 7C, 7N1 or 7N2-1 () at various concentrationsand the attachment of G361 cells was determined. The numbers of cellsattached to these peptides were correlated with the cellular phosphataseactivity which is expressed as absorbance at 410 nm.

FIGS. 3A and 3B show the effect of the active subpeptides of C4 and C7on G361 cell attachment to the rCBD of TS1. To microtiter wellscontaining immobilized rCBD (10 μg/ml), G361 cells were added with (FIG.3A) 4N1-1 (), 7N3-1 (▪), 4N1-1 plus 7N3-1 (▴) and with (FIG. 3B) 4N1(▪), 7N3 () and 4N1 plus 7N3 (▴). The cells were incubated for 1.5 hrin the wells and cell attachment was determined. The effects of thesesubpeptides on cell attachment are expressed as percent relative to themaximum cell attachment for each test where 100% is the number of cellsbinding to rCBD-coated well in the absence of peptide inhibitors.

FIG. 4 shows cell attachment sites in TS1 and LM. Amino acid sequencecomparison between a LM peptide designated LMF9 with 4N-1 and LM 22-2with 7N3 is shown. Amino acid residues which are identical are shown insolid boxes while conservative substitutions are shown in broken boxes.The alignments produced were modified by the addition of a gap in theLMF9 in order to maintain the best alignment.

FIGS. 5A, 5B, 5C and 5D show the effects of soluble peptides of TS1 orLM on G361 cell attachment to immobilized TS1 or LM. (FIG. 5A) In thestandard assay, G361 cells alone (no inhibitors) were added tomicrotiter wells coated with LM at the indicated concentrations. Ininhibition studies, soluble subpeptides were included with G361 cells atthe indicated concentrations. Soluble LM peptides LGTIPG, [SEQ ID NO:9],PGAIPG, [SEQ ID NO:10], YIGSR [SEQ ID NO:11], (▪), LM1 (), and LM2 (▴)were added to (FIG. 5B) LM-coated wells or (FIG. 5D) rCBD-coatedsurface. Soluble TS1 subpeptides 4N1-1 (▪), 7N3-1 (▴), 4N1-1 plus 7N3-1() were tested on (FIG. 5C) LM-coated wells. Control attachment (100%)is that with no added peptides. Data are shown for concentrations ofeach peptide below its solubility limit.

FIGS. 6A, 6B, 6C and 6D show the attachment of four cell types toimmobilized TS-1 and LM. Wells were coated with the indicatedconcentrations of TS1 (▪) or LM (). Assays were performed as describedunder “Methods” hereinafter with (FIG. 6A)—G361 human melanomas; (FIG.6B)—K562 erythroleukemia cells; (FIG. 6C)—HT1080 fibrosarcomas; and(FIG. 6D)—C32 amelanotic melanomas added to the wells.

FIG. 7 is a graphical representation which shows the effect of peptidesfrom TS1 and LM on K562 cell attachment to immobilized rCBD of TS1.Wells were coated with 10 μg/ml of rCBD/well. Prior to addition of K562cells, indicated concentrations of peptide LM1 (▪), LM2 (), 4N-1 (♦),7N3-1 (▴), and 4N1-1 plus 7N3-1 (¤) were added to the wells. Controlattachment (100%) is that with no added peptides.

FIGS. 8A and 8B show conserved cell-binding regions of the CBD of knownTS1 isoforms. In FIG. 8A residues identical among the TS1 sequences areshown in the box. In FIG. 8B, only residues which differ are boxed.References for each sequence are indicated in the detailed descriptionhereinafter.

FIG. 9A shows a region of human and mouse tenascin compared with peptide7N3 of the CBD of TS1. Boxes enclose identical residues. FIG. 9B showsother extracellular proteins that contain sequences homologous to the7N3-1 sequence of TS1, including IKVAV, [SEQ ID NO:12], IRVAV, [SEQ IDNO:13], and VRVAV [SEQ ID NO:14].

The novel binding peptides of this invention can be prepared by knownsolution and solid phase peptide synthesis methods.

In conventional solution phase peptide synthesis, the peptide chain canbe prepared by a series of coupling reactions in which the constituentamino acids are added to the growing peptide chain in the desiredsequence. The use of various N-protecting groups, e.g., thecarbobenzyloxy group or the t-butyloxycarbonyl group (BOC), variouscoupling reagents, e.g., dicyclohexylcarbodiimide or carbonyldimidazole,various active esters, e.g., esters of N-hydroxyphthalimide orN-hydroxy-succinimide, and the various cleavage reagents, e.g.,trifluoroacetic acid (TFA), HCl in dioxane, borontris-(trifluoroacetate) and cyanogen bromide, and reaction in solutionwith isolation and purification of intermediates is well-known classicalpeptide methodology.

The preferred peptide synthesis method follows conventional Merrifieldsolid-phase procedures. See Merrifield, J. Amer. Chem. Soc. 85, 2149-54(1963) and Science 150, 178-85 (1965). This procedure, though using manyof the same chemical reactions and blocking groups of classical peptidesynthesis, provides a growing peptide chain anchored by its carboxyterminus to a solid support, usually cross-linked polystyrene,styrenedivinylbenzene copolymer or p-methylbenz-hydrylamine polymer forsynthesizing peptide amides. This method conveniently simplifies thenumber of procedural manipulations since removal of the excess reagentsat each step is effected simply by washing the polymer.

Further background information on the established solid phase synthesisprocedure can be had by reference to the treatise by Stewart and Young.“Solid Phase Peptide Synthesis,” W. H. Freeman & Co., San Francisco,1969, and the review chapter by Merrifield in Advances in Enzymology 32,pp. 221-296, F. F. Nold, Ed., Interscience Publishers, New York, 1969;and Erickson and Merrifield, The Proteins, Vol. 2, p. 255 et seq. (ed.Neurath and Hill), Academic Press, New York, 1976.

In order to illustrate the invention in further detail, the followingspecific laboratory examples were carried out with the results asindicated. Although specific examples are thus illustrated herein, itwill be appreciated that the invention is not limited to these specificexamples or the details therein. It will further be understood that thenovel peptides of this invention are not limited to any particularmethod of synthesis.

EXAMPLES Materials and Methods

The generation and characterization of mAB C6.7 and its epitopelocalization to peptide C7 within the CBD of TS1 were carried out byconventional published procedures (Dixit et al., 1985, Kosfeld et al.,1991). All cell lines used are conventional and were obtained bypurchase from ATCC: human melanoma G361 (CRL 1424), K562 humanerythroleukemia cells (ATCC CCL 241), HT-1080 human fibrosarcomas (ATCCCCL 121) and C32 amelanolic human melanoma (ATTC CRL 1585). Cells werecultured in RPM1 1640 medium supplemented with 10% fetal calf serum(FCS) at 5% CO₂ (Roberts et al., 1987). All reagents were obtained fromSigma Chemical Co. (St. Louis, Mo.) unless specified otherwise.

The rCBD of TS1—The rCBD was expressed by conventional procedures aspreviously described (Kosfeld et al., 1991). The rCBD was found in thesoluble fraction of bacterial lysates, and was purified bychromatography on a column of Q-Sepharose “fast flow” (Pharmacia FineChemicals, Piscataway, N.J.) equilibrated with 50 mM Tris-HCl, pH 7.5,0.15 M NaCl (TBS). The column was eluted with a linear gradient of NaCl(0.15-1 M) in the Tris buffer. Fractions with the highest concentrationof the protein A-CBD fusion protein were identified by SDS-PAGE using7.5% acrylamide gels, followed by staining with Coomassie Blue andwestern blotting using alkaline phosphatase-conjugated IgG to locatebands containing the protein A moiety. The rCBD fractions were dialyzedagainst PBS and stored at −70° C. until used. It is known that theprotein A moiety has no attachment activity for these cells.

Peptide Synthesis—Peptides whose sequences correspond to portions ofpeptide C4 (FIG. 1A) or C7 (FIG. 2A) of the CBD of TS1 were synthesizedand purified by conventional procedures as described previously (Prateret al., 1991). Briefly, peptides were made on an Applied BiosystemsModel 3804 solid phase peptide synthesizer. The resultant peptides werecleaved and deblocked by Immuno-dynamics (San Diego, Calif.) andpurified by reversed phase HPLC (Waters) using acetonitrile/0.1% TFAsolvent systems. Purity was tested with analytical HPLC. Primarystructures of peptides were confirmed by amino acid composition on aBeckman model 6300 amino acid analyzer, and by sequencing with anApplied Biosystems model 477A sequencer. All peptide preparations weretested for cytotoxicity on G361 cells and were not toxic at theconcentrations employed in these tests.

Cell Adhesion Assay—Cell adhesion was performed in 96-well plates byconventional procedures as previously described (Prater et al., 1991;Kosfeld and Frazier, 1992). Synthetic peptides were solubilized with 6 Nguanidine HCl (GnHCl) because some hydrophobic peptides were insolubleat concentrations approaching 1 mM in TBS. The peptides were coated ontoplastic 96 well plates (Nunc Immuno Plate Maxisorp) by incubating 50 μlof peptide solutions per well at the indicated concentrations. Afterincubating overnight at room temperature, wells were rinsed with TBS andblocked with 10 mg/ml bovine serum albumin (BSA) for 30 minutes at roomtemperature. Peptides were also coupled to cyanogen bromide-activatedSepharose 4B (Pharmacia) according to the manufacturer's instructions.Approximately 2 mg of peptide was used per ml of the Sepharose. Theefficiency of coupling of all peptides was greater than 90% as judged byUV absorbance. The peptide-coated beads were then blocked with BSA asabove. IgG coated beads were also prepared for use as controls.

After blocking, cells were allowed to attach for 1 hr at 37° C. tosubstrate-coated wells or beads. In some tests, inhibition wasdetermined by adding peptide inhibitors of cell attachment to each wellalong with the cells. After removal of non-adhering cells, cellsattached to immobilized substrates were quantitated with endogenouscellular phosphatase activity which is measured by absorption at 410 nmas previously described (Prater et al., 1991). Each assay was carriedout in duplicate and each peptide was tested in 3 separate tests at 3 ormore different concentrations.

Peptide-BSA conjugates—In addition to the free peptides, peptidesconjugated to protease-free BSA (Humphries et al., 1987) were also usedin the cell binding assay. Peptides were synthesized with an N-terminalcysteine for covalent coupling to BSA via the heterobifunctionalcrosslinking reagent N-succinimidyl 3-(2-pyridyldithio) propionate(SPDP). BSA was activated at room temperature with SPDP at a molar ratioof 50:1 (SPDP:BSA) for 30 min. Unreacted SPDP was separated fromderivatized BSA by gel filtration on a Sephadex G-25SF columnequilibrated with PBS. The activated BSA was then added to dry peptideat a molar ratio of 1:20 (BSA:peptide). The reactants were mixedovernight at room temperature and unconjugated peptides removed bydialysis against PBS, pH 7.4. Peptide-BSA conjugates were stored frozenat −20° C. until used.

Results

The amino acid sequences of the subpeptides synthesized from C4 areshown in FIG. 1A. As a first step to locate the essential amino acidsequence(s) within C4, two peptides with overlapping sequences, 4N and4C, were synthesized and their activities assessed using the cellattachment assay. The results in FIG. 1B show that peptide 4N, but not4C, displayed high activity. In order to further locate the activesequence(s) within 4N, three shorter overlapping peptides spanning 4N(FIG. 1A) were tested. Peptide 4N1,Arg-Phe-Tyr-Val-Val-Met-Trp-Lys-Gln-Val-Thr-Gln-Ser [SEQ ID NO:1],showed significantly greater cell binding activity than peptide 4N2.4N1-1, the amino-terminal sequence of 4N1, retained 60% of the maximumactivity of 4N1. These results indicate the importance of the sequencecommon to both active peptides, 4N1 and 4N1-1, the novel octapeptideArg-Phe-Tyr-Val-Val-Met-Trp-Lys [SEQ ID NO:2].

The amino acid sequences of the C7 subpeptides is shown in FIG. 2A andtheir cell attachment activities in FIG. 2B. First, two peptides, 7N and7C, which together represent the entire length of C7 were synthesized.7C actually extends beyond the C terminus of C7 (arrow in FIG. 2). Theamino-terminal peptide, 7N, showed significant cell attachment activitywhile the COOH-terminal peptide, 7C, exhibited none. Next, 7N wasdivided into three overlapping subpeptides designated 7N1, 7N2, and 7N3.Of these, only 7N2 and 7N3 had significant activity, the activity of 7N2being less than half of that of 7N3,Phe-Ile-Arg-Val-Val-Met-Tyr-Glu-Gly-Lys-Lys [SEQ ID NO:4]. Based on thisresult, 7N3 was further dissected into two pentapeptides, 7N3-1 and7N3-2. Peptide 7N2 was also divided into two smaller subpeptides, 7N2-1and 7N2-2. The 7N2-2 sequence contains the COOH-terminal sequence of the7N2 and the amino-terminal sequence of the 7N3-1. The maximal cellattachment activity of 7N3-1 was comparable to that of 7N3, and nearlyas high as that of 7N and the parent peptide C7. Peptides 7N2-1, 7N2-2and 7N3-2 on the other hand, exhibited negligible attachment-promotingactivities. These results localize the highest cell attachmentactivities to peptides containing the central region of C7 such as 7N,7N3 and 7N3-1, indicating that the critical residues for activity lie inthe 7N3-1 sequence. Thus both the aforesaid active octapeptide sequencefrom C4, and the pentapeptide sequence from C7, Ile-Arg-Val-Val-Met [SEQID NO:5], contain the VVM sequence, the only sequence shared by C4 andC7.

To ascertain that the activities of the peptides are not a function oftheir association with the plastic surface, peptides linked to BSA orSepharose beads were also used in cell binding assays. Again the samepeptides that are shown to be active in FIGS. 1 and 2 also promotesubstantial cell attachment when conjugated to BSA and then coated onplastic wells, or when covalently attached to Sepharose beads. Theseobservations confirm the aforesaid sequences 4N1-1 from C4 and 7N3-1from C7 as the primary determinants of the activity of the CBD of TS1.

To be sure that the active peptides from C4 and C7 contain the sequencesthat are relevant for attachment of cells to the CBD, the shorter, moresoluble active peptides were tested as soluble inhibitors of the bindingof G361 cells to rCBD immobilized on plastic wells. The short peptide,7N3-1, in contrast to its larger homolog 7N3, is highly soluble, whichmakes it possible to test it at the high concentrations often requiredfor inhibition of cell attachment. In contrast, 4N1-1 and 4N1, theactive subpeptides for C4, are insoluble at concentrations higher than0.2 mM and thus must be tested as soluble inhibitors at concentrationsbelow this solubility limit. It should be noted that inclusion ofpeptides in the cell attachment assay at these concentrations had noadverse effects on the cells. The ability of the active subpeptides fromC4 and C7 to inhibit G361 melanoma cell adhesion to rCBD is shown inFIG. 3. The results of this complementary bioassay confirmed those ofthe direct cell adhesion assays (FIGS. 1 and 2). It was found thatpeptides 4N1 and 7N3 interfere with the attachment of G361 cells torCBD-coated surfaces by about 30% and 50%, respectively (FIG. 3B) whilethe shorter peptides 4N1-1 and 7N3-1 were inhibitory by 30% and 25%,respectively (FIG. 3A). The inhibition was dose-dependent at peptideconcentrations <0.1 mM for 4N1 and 7N3 and <0.2 mM for 4N1-1 and 7N3-1.7N3-1 tested at 2 mM exhibits no significant increase in inhibitoryeffect compared to that at 0.2 mM. Thus the shortest active subpeptidesfrom C4 or C7, when added individually to cells, only partially inhibitcell attachment to the rCBD. A combination of these subpeptides however,had a synergistic effect as shown in the triangles in FIGS. 3A and 3B.Each peptide is present at ½ the indicated concentration. In the case of4N1 plus 7N3, the inhibition was nearly complete (87%). In contrast, theeffect of 4Nl-1 plus 7N3-1 was less than that of the longer 4N1 plus 7N3peptides, suggesting that some active amino acid residues might bemissing from the shorter peptides. In contrasting FIG. 3A to 3B, theactivity exhibited by 4N1-1 and 4N1 was comparable while peptide 7N3-1was not as active as 7N3. This suggests that 7N3-1 does not contain allof the active residues. To evaluate the contribution of adjacentsequences to activity, peptide 7N2-2 which contained the N-terminalsequence of 7N3-1, and 7N3-1 were examined in inhibition studies. Whileless inhibitory than 7N3-1, both peptides showed significant inhibitoryactivity, suggesting the importance of adjacent sequences in modifyingthe activity of the 7N3-1 peptide. Peptides that are distant from theactive sequences of C4 and C7 (4C, 4N2, 7N1) showed no inhibition ofcell attachment to the rCBD, indicating that the inhibition is specificand not due to cytotoxic effects of the peptide preparations.

In the course of these inhibition tests, a shorter peptide,Arg-Phe-Tyr-Val-Val-Met [SEQ ID NO:3], derived from 4N1-1 (designated4N1-2) was tested. This hexapeptide had little or no activity in thedirect cell adhesion assays (FIG. 1) yet in the inhibition assays was aspotent as the longer 4N1-1. This may represent a case of a peptide thateither binds very poorly to the plastic wells or, when bound, assumes aconfiguration on the plastic that prevents it from interacting withcellular receptors. Peptides which alter the sequence of 7N3-1 were alsosynthesized to test the importance of the isoleucine, arginine andmethionine residues in binding to cellular receptors. These peptides,Gly-Arg-Val-Val-Met [SEQ ID NO:6], Ile-Glu-Val-Val-Met [SEQ ID NO:7] andIle-Arg-Val-Val-Gly [SEQ ID NO:8] were tested as inhibitors of celladhesion to the rCBD. All were inactive.

The aforesaid octapeptide 4N1-1 and pentapeptide 7N3-1 were thusidentified as important sequences for the cell attachment activity ofthe TS1 CBD. Further, the hexapeptide 4N1-2 is active as an inhibitor ofcell binding to the rCBD. These peptides share the tripeptide VVM andboth contain an arginine upstream. Search was then made for sequencesrelated to these peptides in other cell adhesion and ECM proteins(Yamada, 1991). This search revealed two peptides from laminin (LM)having sequences similar to 4N1 and 7N3. The sequence homology betweenthese peptides is shown in FIG. 4. The peptide designated LMF9, from theF9 fragment of LM (Skubitz et al., 1990), shares 5 identical residuesand 2 conservative substitutions with 4N1. LM22-2, from peptide PA22.2of LM (Tashiro et al., 1989), has sequence homology (not identity) with7N3, particularly within the LM-2 active region of the LM peptide. Twopeptides modeled on these residues of LM (FIG. 4) were synthesized andtested as substrates for attachment of G361 cells. LM-1 (the LMF9 analogof TS1 4N1-1) had little cell attachment activity while LM-2 (the PA22-2analog of TS1 7N3-1) bound cells but to a lesser degree than the 7N3-1.These results were confirmed using peptides linked to BSA and Sepharosebeads.

The ability of these peptide homologs to substitute for each other ininhibiting G361 cell binding to the TS1 rCBD or LM-coated surfaces (FIG.5) was also tested. G361 cells bound to LM in a concentration-dependentmanner (FIG. 5A) and this attachment activity was inhibited by LM1(21%), LM2 (28%) (FIG. 5B), 4N1-1 (32%) and 7N3-1 (50%) (FIG. 5C). LM1and LM2 also inhibited cell binding to the CBD of TS1, 15% and 34%,respectively (FIG. 5D). Quantitatively, LM peptides were less effectivethan their TS1 homologs. Other adhesive peptides of LM were also testedas inhibitors of the CBD and LM but showed no detectable activity (FIGS.5B and 5D). These results suggest that TS1 and LM may be able to share acommon receptor.

To further address this issue, several cell types and lines were testedfor attachment to LM and TS1 to see if any cells attached to one proteinbut not the other. FIG. 6 shows attachment to TS1 and LM of four celltypes. These include G361 human melanomas (FIG. 6A). K562 humanerythroleukemia cells (FIG. 6B), HT1080 human fibrosarcomas (FIG. 6C)and C32 human melanoma (FIG. 6D). It is of interest to note that C32 andK562 cells attached to TS1 much more avidly than to LM while G361 andHT1080 cells bound well to both proteins. The K562 cells also bound wellto the rCBD and its active sequences. Next the effect of 4N1-1, 7N3-1and their LM homologs on K562 cell attachment to the rCBD was tested.Since these cells demonstrated specific attachment to TS1 and not to LM,no LM receptor should contribute to the binding of the TS1 CBD. Asfollows from the previous results, the TS1 peptides showed significantinhibitory activity (36% for 4N1-1, 28% for 7N3-1) while the LM peptidesexhibited an undetectable (LM1) or a lower (19% for LM2) level ofinhibition (FIG. 7). In agreement with previous results, the combinationof 4N1-1 and 7N3-1 increased the inhibition of rCBD-mediated cellattachment (45%). The TS1 peptides thus, are better competitors than theLM peptides in the absence of a LM receptor interaction. K562 cellstherefore provide a valuable model in which only the cellularinteractions with TS1 can be evaluated. Thus it appears that a receptorexists which binds the rCBD of TS1 with a high degree of specificitywhich excludes an interaction with LM.

Based on the assumption that the active sequences in the CBD arecritical for cell attachment, it would appear that these residues wouldbe conserved among different species. FIG. 8 shows the alignment of thehomologs of peptides 7N3 and 4N1 from all known isoforms and species ofTS. While peptide 7N3 is highly conserved only in human and mouse TS1,similar residues are retained in human, mouse and chicken TS2. In mouseTS3 and in bovine and rat COMP, positively charged residues arerearranged. Peptide 4N1, however shows an extremely high degree ofconservation in all species and isoforms of TS sequenced to date. Infact within the region of the shortest active peptide, 4N1-1, there areonly two substitutions in all known sequences. This conservation ofsequence indicates that this is a region of the TS1 CBD responsible fora critical function that may be retained through evolution and invarious TS isoforms.

The sequences 4N1-1, 7N3 and 7N3-1 were used as probe sequences in acomputer search of all available data bases. No perfect matches werefound for the octapeptide 4N1-1 sequence or its shorter inhibitoryhexapeptide form 4N1-2. Similarly, the 7N3-1 pentapeptide sequenceappears to be unique to TS1 isoforms among extracellular proteins.However, searching for homologs of the 7N3 sequences revealed that humanand mouse tenascin contain a related sequence shown in FIG. 9. Sinceevidence has been obtained (above) that the LM sequence LM-2 can atleast partially substitute for the TS1 sequence 7N3-1, a search was madefor combinatiorially interchanged sequences. As shown in FIG. 9B,several interesting extracellular proteins contain related sequencesincluding human and mouse LM, human and porcine von Willebrand factor,porcine factor VIII and rat α-2-macroglobulin. These proteins have beensingled out because they interact with cells through receptors, some ofwhich have not yet been identified. It is believed that the TS1homologous sequences represent occurrences of a similar receptor bindingmotif in these proteins. These novel sequences are as follows:

Ile Arg Val Ala Val [SEQ ID NO:13] and                 5 Val Arg Val AlaVal [SEQ ID NO:14].                 5

Amino acids are shown herein either by standard one letter or threeletter abbreviations as follows:

Abbreviated Designation Amino Acid A Ala Alanine C Cys Cysteine D AspAspartic Acid E Glu Glutamic Acid F Phe Phenylalanine G Gly Glycine HHis Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M MetMethionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg ArginineS Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y TyrTyrosine

References shown in parentheses herein are appended at the end.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

References

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47 13 amino acids amino acid linear peptide 1 Arg Phe Tyr Val Val MetTrp Lys Gln Val Thr Gln Ser 1 5 10 8 amino acids amino acid linearpeptide 2 Arg Phe Tyr Val Val Met Trp Lys 1 5 6 amino acids amino acidlinear peptide 3 Arg Phe Tyr Val Val Met 1 5 11 amino acids amino acidlinear peptide 4 Phe Ile Arg Val Val Met Tyr Glu Gly Lys Lys 1 5 10 5amino acids amino acid linear peptide 5 Ile Arg Val Val Met 1 5 5 aminoacids amino acid linear peptide 6 Gly Arg Val Val Met 1 5 5 amino acidsamino acid linear peptide 7 Ile Glu Val Val Met 1 5 5 amino acids aminoacid linear peptide 8 Ile Arg Val Val Gly 1 5 6 amino acids amino acidlinear peptide 9 Leu Gly Thr Ile Pro Gly 1 5 6 amino acids amino acidlinear peptide 10 Pro Gly Ala Ile Pro Gly 1 5 5 amino acids amino acidlinear peptide 11 Tyr Ile Gly Ser Arg 1 5 5 amino acids amino acidlinear peptide 12 Ile Lys Val Ala Val 1 5 5 amino acids amino acidlinear peptide 13 Ile Arg Val Ala Val 1 5 5 amino acids amino acidlinear peptide 14 Val Arg Val Ala Val 1 5 30 amino acids amino acidlinear peptide 15 Arg Phe Tyr Val Val Met Trp Lys Gln Val Thr Gln SerTyr Trp Asp 1 5 10 15 Thr Asn Pro Thr Arg Ala Gln Gly Tyr Ser Gly LeuSer Val 20 25 30 18 amino acids amino acid linear peptide 16 Arg Phe TyrVal Val Met Trp Lys Gln Val Thr Gln Ser Tyr Trp Asp 1 5 10 15 Thr Asn 10amino acids amino acid linear peptide 17 Lys Gln Val Thr Gln Ser Tyr TrpAsp Thr 1 5 10 16 amino acids amino acid linear peptide 18 Trp Asp ThrAsn Pro Thr Arg Ala Gln Gly Tyr Ser Gly Leu Ser Val 1 5 10 15 30 aminoacids amino acid linear peptide 19 Arg Trp Arg Leu Ser His Arg Pro LysThr Gly Phe Ile Arg Val Val 1 5 10 15 Met Tyr Glu Gly Lys Lys Ile MetAla Asp Ser Gly Pro Ile 20 25 30 22 amino acids amino acid linearpeptide 20 Arg Trp Arg Leu Ser His Arg Pro Lys Thr Gly Phe Ile Arg ValVal 1 5 10 15 Met Tyr Glu Gly Lys Lys 20 11 amino acids amino acidlinear peptide 21 Arg Trp Arg Leu Ser His Arg Pro Lys Thr Gly 1 5 10 9amino acids amino acid linear peptide 22 Arg Pro Lys Thr Gly Phe Ile ArgVal 1 5 6 amino acids amino acid linear peptide 23 Arg Pro Lys Thr GlyPhe 1 5 6 amino acids amino acid linear peptide 24 Lys Thr Gly Phe IleArg 1 5 5 amino acids amino acid linear peptide 25 Tyr Glu Gly Lys Lys 15 13 amino acids amino acid linear peptide 26 Tyr Glu Gly Lys Lys IleMet Ala Asp Ser Gly Pro Ile 1 5 10 13 amino acids amino acid linearpeptide 27 Arg Phe Tyr Val Val Met Trp Lys Gln Val Thr Gln Ser 1 5 10 12amino acids amino acid linear peptide 28 Arg Tyr Val Val Leu Pro Arg ProVal Cys Phe Glu 1 5 10 7 amino acids amino acid linear peptide 29 ArgTyr Val Val Leu Pro Arg 1 5 11 amino acids amino acid linear peptide 30Phe Ile Arg Val Val Met Tyr Glu Gly Lys Lys 1 5 10 10 amino acids aminoacid linear peptide 31 Ser Ile Lys Val Ala Val Ser Ala Asp Arg 1 5 10 11amino acids amino acid linear peptide 32 Phe Ile Arg Val Val Met Tyr GluGly Lys Lys 1 5 10 11 amino acids amino acid linear peptide 33 Tyr IleArg Val Val Met Tyr Glu Gly Lys Lys 1 5 10 11 amino acids amino acidlinear peptide 34 Tyr Ile Arg Val Leu Val His Glu Gly Lys Gln 1 5 10 11amino acids amino acid linear peptide 35 Tyr Met Arg Val Leu Val His GluGly Lys Gln 1 5 10 11 amino acids amino acid linear peptide 36 Leu IleLys Val Leu Val Tyr Glu Gly Lys Gln 1 5 10 11 amino acids amino acidlinear peptide 37 Tyr Ile Arg Val Lys Leu Tyr Glu Gly Pro Gln 1 5 10 11amino acids amino acid linear peptide 38 Tyr Ile Arg Val Arg Phe Tyr GluGly Pro Glu 1 5 10 11 amino acids amino acid linear peptide 39 Arg PheTyr Val Val Met Trp Lys Gln Val Thr 1 5 10 11 amino acids amino acidlinear peptide 40 Arg Phe Tyr Val Val Met Trp Lys Gln Val Thr 1 5 10 11amino acids amino acid linear peptide 41 Arg Phe Tyr Val Val Met Trp LysGln Val Thr 1 5 10 11 amino acids amino acid linear peptide 42 Arg PheTyr Val Val Met Trp Lys Gln Val Thr 1 5 10 11 amino acids amino acidlinear peptide 43 Arg Phe Tyr Val Leu Met Trp Lys Gln Val Thr 1 5 10 11amino acids amino acid linear peptide 44 Arg Phe Tyr Val Val Met Trp LysGln Thr Glu 1 5 10 11 amino acids amino acid linear peptide 45 Ser PheTyr Val Val Met Trp Lys Gln Met Glu 1 5 10 11 amino acids amino acidlinear peptide 46 Phe Ile Arg Val Val Met Tyr Glu Gly Lys Lys 1 5 10 12amino acids amino acid linear peptide 47 Phe Ile Arg Val Phe Ala Ile LeuGlu Asn Lys Lys 1 5 10

What is claimed is:
 1. A peptide that binds to the thrombospondin 1receptor and having the sequence Ile Arg Val Ala Val [SEQ ID NO:13]. 2.A peptide that binds to the thrombospondin 1 receptor and having thesequence Val Arg Val Ala Val [SEQ ID NO:14].